Culturing bacteria on agar plates is a fundamental practice in microbiology, allowing scientists to study microorganisms, isolate specific types, and quantify their presence. An agar plate consists of a Petri dish containing a solid growth medium. This medium provides a controlled environment with necessary nutrients for microbial growth.
Preparing for Plating
Before bacteria can be spread onto an agar plate, preparatory steps ensure a clean environment for microbial growth. Maintaining sterility prevents contamination from unwanted microorganisms, which are naturally present on surfaces and in the air. Working near a Bunsen burner flame helps reduce airborne contaminants, and a laminar flow hood provides a sterile filtered air workspace.
Agar, a polysaccharide, serves as the solidifying agent in the growth medium, providing a stable surface for bacterial colonies. Growth media are formulated with nutrients like proteins, carbohydrates, and minerals, tailored to support the specific bacterial species being cultured. These media are prepared by mixing ingredients, heating to dissolve the agar, and then sterilizing, often using an autoclave at 121°C for about 15-25 minutes.
After sterilization, the molten agar medium is cooled to approximately 45-50°C before being poured into sterile Petri dishes. Once solidified, the plates are ready for inoculation. The bacterial sample, or inoculum, is often prepared as a liquid culture or a diluted suspension to achieve an appropriate concentration for spreading. Common sterile tools include inoculation loops, spreaders, and pipettes.
Common Spreading Methods
Several techniques are employed to spread bacteria on agar plates, each serving a distinct purpose.
Streak Plating
The streak plating method is widely used for isolating individual bacterial colonies from a mixed sample. This technique progressively dilutes the bacterial inoculum across the agar surface to achieve single cells that can grow into distinct colonies.
A common approach involves a four-quadrant streaking pattern. A sterile inoculation loop picks up bacterial culture and spreads it across the first quadrant. The loop is sterilized, then used to drag cells from the first quadrant into the second, further diluting the sample. This process repeats for the third and fourth quadrants, resulting in increasingly diluted areas where isolated colonies are expected to grow.
Spread Plating
Spread plating is primarily used for quantifying viable bacterial cells in a liquid sample. A measured volume of bacterial suspension is pipetted onto the center of a solidified agar plate. A sterile spreader then evenly distributes the liquid inoculum across the entire agar surface. The plate is rotated during spreading to ensure uniform distribution. This method allows for the enumeration of colony-forming units (CFUs), providing a count of living bacteria in the original sample.
Pour Plating
Pour plating involves mixing the bacterial sample directly with molten agar before it solidifies. A measured volume of bacterial suspension is added to a sterile Petri dish, then molten agar is poured into the dish. The dish is gently swirled to mix the sample within the agar. Once solidified, the plate is incubated. This technique allows for the growth of both aerobic and anaerobic microorganisms, as colonies can develop throughout the depth of the agar medium. Pour plating is also commonly used for quantifying bacterial populations, particularly in food and water testing.
Incubation and Colony Growth
After spreading bacteria onto agar plates, the inoculated plates are placed in an incubator to provide optimal conditions for bacterial growth. Temperature is a factor, with most human pathogens growing well between 35°C and 37°C, while some environmental bacteria and fungi may require temperatures between 20°C and 30°C. The incubation period typically ranges from 18 to 48 hours for many common bacteria.
Atmospheric conditions are also considered. Plates can be incubated in ambient air for aerobic organisms, or in anaerobic jars or chambers for those requiring an oxygen-free environment. During incubation, individual bacterial cells multiply repeatedly, forming visible masses called colonies. Each colony represents a cluster of millions of cells originating from a single bacterium.
Once colonies have grown, they are observed for characteristics known as colony morphology. This includes assessing their size, shape, elevation, edge, surface texture, opacity, and color. These visual cues provide initial information that can help identify and differentiate bacterial species.
Safe Handling and Disposal
Working with bacterial cultures necessitates strict adherence to safety protocols to protect individuals and prevent environmental contamination. Personal protective equipment (PPE) forms a barrier against potential hazards, including laboratory coats to protect clothing and skin, gloves for hand protection, and eye protection such as safety glasses or goggles.
Aseptic technique minimizes the transfer of microorganisms. This involves working in a disinfected area and sterilizing tools between uses.
Proper disposal of contaminated materials is also important. Used agar plates, pipettes, and other materials that have come into contact with bacteria are considered biohazardous waste. These items must be sterilized, typically by autoclaving, before being disposed of. Autoclaving uses high-pressure steam at 121°C for a recommended period, often 60 minutes for waste, to deactivate pathogens. Following any laboratory work, thorough handwashing with soap and water is important.